M-CSF is a protein which exhibits the spectrum of activity understood in the art for M-CSF, also known as CSF-1, i.e., when applied to the standard in vitro colony stimulating assay of Metcalf, D., J. Cell. Physiol. (1970) 76:89 as modified by Ralph et al., Blood (1986) 68:633, it is capable of stimulating the formation of primarily macrophage colonies. Native M-CSF is a glycosylated dimer; dimerization is reported to be necessary for activity as the M-CSF monomer is not active in the Metcalf or Ralph colony stimulating assays or various other in vitro bioactivity assays (Das, S. K. et al., 1981, Blood 58:630-641; Das, S. K. et al., 1982, J. Biol. Chem. 257:13679-13681; Stanley, E. R. et al., 1977, J. Biol. Chem. 52:4305-4312, Halenbeck, R. et al., 1989Bio/Technology, 7:710-715). The term "M-CSF" refers to proteins that have M-CSF activity in the assays described above and are substantially homologous to the native sequence. Examples of M-CSF sequences, a discussion of various deletion mutants, and processes for bacterial production are shown in U.S. Pat. Nos. 4,847,201 and 4,929,700 which are hereby incorporated by reference in their entireties. Generally, the term "M-CSF" can include M-CSF monomer, M-CSF dimer, or M-CSF refractile body. However, more specific terms, i.e., M-CSF monomer, will be used whenever possible. "M-CSF monomer" refers to a polypeptide comprising an amino acid sequence of a single subunit of an active M-CSF dimer. The amino acid sequence need not be identical to the M-CSF monomer sequences found in nature. Instead, the M-CSF monomer sequences may be routants and/or fragments of the naturally occurring M-CSF monomer sequences. However, M-CSF monomers can be folded and oxidized to form with another M-CSF monomer an active M-CSF dimer. "M-CSF dimer" refers to the biologically active dimer of M-CSF monomers. Biological activity of M-CSF dimers can be measured in any of the assays described above, Metcalf, Ralph et al., Das et al. (1981 & 1982), Stanley et al., and Halenbeck et al., supra. The size the M-CSF dimer can be measured by standard protein gels as described by Sambrook et al., infra.
To purify M-CSF dimer from human sources, such as urine, is very cumbersome. However, recombinant DNA technology has facilitated production of M-CSF dimer, and the pharmaceutical industry can bring such drugs to the public more efficiently without the need for expensive and dangerous human products. Expressing the protein is only part of the process of making a pharmaceutical. The protein must also be purified, refolded, if necessary, and formulated for storage. Methods for producing, purifying, refolding, formulating, and lyophilizing M-CSF dimer are described, for example, in U.S. Pat. No. 4,929,700 and WO89/10407.
As stated above, U.S. Pat. No. 4,929,700 discloses a process to produce and recover M-CSF from bacterial cells in a properly refolded form. U.S. Pat. No. 4,929,700 discloses the following process steps of: fermentation and harvest of the bacterial cells; primary recovery of intracellular pellets or aggregates containing M-CSF monomer, called "M-CSF refractile bodies" (M-CSF RB); solubilization and denaturation of M-CSF RB; refolding & oxidation; column chromatography and purification; and formulation. U.S. Patent '700 is relevant as background, because the present invention is an advantageous modification of this earlier process.
The present invention can purify and refold M-CSF monomers into M-CSF dimers from M-CSF RB which vary greatly in their physical characteristics. Generally, refractile bodies are porous aggregates of proteins that are produced by such organisms as E. coli or yeast. Other cell components, such as carbohydrates, may become associated with refractile bodies. The composition of these components can change depending on the health of the cells producing the refractile bodies, the growth conditions, or the cell harvesting conditions. For example, if the bacterial cells begin producing heat shock proteins, different cell metabolites may associate with the refractile bodies, and consequently, the refractile bodies may exhibit different physical characteristics. Also, when the bacterial cells are lysed, the media components or cellular components, lipids and DNA, for example, may become associated with the refractile bodies. Further, during front-end purification procedures, contaminants may associate with the harvested refractile bodies. Thus, such changes in the size, density, hydrophobicity, and other characteristics of the refractile bodies can create bottlenecks in the downstream processing and result in eventual loss of the desired proteins from the refractile bodies.
Once the protein is produced, refolded, and purified, formulation and lyophilization procedures are critical for long term maintenance of protein stability. Typical lyophilization schemes comprise a freezing step, a primary drying step, and a secondary drying step.
The formulation is frozen in order to:
(1) freeze the protein; PA1 (2) freeze the unwanted water; and PA1 (3) form a matrix, to facilitate reconstitution of the protein. PA1 (1) removing as many M-CSF aggregates as possible before formulation; and PA1 (2) adding a polyoxyethylenic non-ionic surfactant to the formulation. PA1 1. Fermentation & Harvest; PA1 2. Primary Recovery; PA1 3. Solubilization & Denaturation of M-CSF RB; PA1 4. Refolding & Oxidation; and PA1 5. Column Chromatography, & Purification. PA1 exposing the harvested M-CSF RB to an effective amount of a guanidine salt to solubilize and denature the harvested M-CSF RB to produce M-CSF monomer mixture; and PA1 dialyzing the M-CSF monomer mixture to remove the contaminants, as measured by absorbance at 280 nm (OD.sub.280 or A.sub.280), that prevent folding of M-CSF monomers. PA1 a) freezing the liquid M-CSF formulation, comprising M-CSF dimer, an effective concentration of mannitol and an effective concentration of an amorphous protectant, to form a frozen product comprising an effective amount of crystallized mannitol; PA1 b) drying the frozen product by raising the product temperature to a final product temperature without a constant drying period under subatmospheric pressures and maintaining the final product temperature.
During the freezing step, the formulation may undergo several temperature shifts to freeze and to properly crystallize the formulation components. See Williams et al., J. Parent. Sci. Tech. 38(2): 48-59 (1984), at page 49, bottom right column. These shifts in temperature are performed at normal atmospheric pressures, and thus, the formulation is not being dried during these temperature shifts.
The formulation is dried in two steps according to known lyophilization procedures. During both primary and secondary drying, the chamber pressure is reduced to force the water to proceed directly from solid to gas phase, i.e., sublimate. The primary drying begins after the formulation is frozen, and most of the water is removed by this step. During primary drying, the pressure in the sample chamber is reduced, and the shelf temperature of the lyophilizer is raised and held constant at a primary drying temperature. The shelf temperature is held constant to allow the product temperature to equilibrate with the shelf temperature as shown in FIG. 1 on page 49 of Williams et al., supra. The water vapor is discharged into the condenser of the lyophilizer, which re-freezes the vapor.
After primary drying is completed, again under reduced chamber pressure, the shelf temperature is raised to a secondary drying temperature and then held constant. Again, the shelf temperature is held constant to allow the product temperature to equilibrate with the shelf temperature. During secondary drying, water which is tightly bound to the product is removed.
One general formulation and lyophilization procedure for M-CSF dimer is described in WO89/10407 by Morris et al. This application relates to specific methods to reduce the number of aggregates of M-CSF after lyophilization. Morris et al., supra describes a method to circumvent such aggregation by:
Thus, WO89/10407 provides no motivation to modify known lyophilization procedures, but merely directs those skilled in the art to two pages from Remington's Pharmaceutical Sciences and the lyophilization procedure in Example 2 of the Morris et al. application. Neither citations suggest any innovation to known lyophilization procedures.